Methods of nucleic acid target capture

ABSTRACT

Methods for efficiently capturing a target nucleic acid from a sample by using a mixture that contains a capture probe specific for the target nucleic acid, the target nucleic acid, and a denaturant chemical, which mixture is incubated at elevated temperature for a short time, are disclosed. Compositions that include a capture probe that specifically binds to a target nucleic acid and a denaturant chemical, which when mixed with the target nucleic acid and incubated at elevated temperature for a short time, promote efficient hybridization of the capture probe and target nucleic acid are disclosed.

RELATED APPLICATION

This application is a divisional of application Ser. No. 11/429,304,filed May 5, 2006, which claims the benefit under 35 U.S.C. 119(e) ofprovisional application No. 60/678,507, filed May 6, 2005, which isincorporated by reference herein.

FIELD OF THE INVENTION

The disclosed compositions and methods relate to molecular biology, moreparticularly to the isolation of nucleic acids from complex mixturessuch as samples by using a nucleic acid oligomer specific for the targetnucleic acid and a denaturant chemical in the mixture.

BACKGROUND OF THE INVENTION

Many molecular biology procedures such as in vitro amplification and invitro hybridization of nucleic acids require some preparation orpurification of the nucleic acids to make them effective in thesubsequent procedure. Methods of nucleic acid purification have beendeveloped that are non-specific and isolate all nucleic acids present ina sample, or isolate different types of nucleic acids based on physicalcharacteristics, or isolate specific nucleic acids from a sample. Manynucleic acid isolation methods involve complicated procedures or use ofharsh chemicals, and require a long time to complete. There remains aneed for a simple, efficient, and fast method to separate a nucleic acidof interest from other sample components.

SUMMARY OF THE INVENTION

A method is disclosed for isolating a target nucleic acid of interestfrom a sample, including the steps of mixing a sample containing atarget nucleic acid with a capture probe that hybridizes specifically toa target sequence in the target nucleic acid in a solution phase thatcontains a denaturant chemical and an immobilized probe that bindsspecifically to the capture probe, to provide a reaction mixture,incubating the reaction mixture at a first temperature in a range ofabout 60° C. to 95° C. for about 15 minutes or less, incubating thereaction mixture at a second temperature in a range of about 25° C. to42° C. for about 20 minutes or less, thereby forming a hybridizationcomplex made up of the capture probe hybridized specifically to thetarget nucleic acid and the immobilized probe bound specifically to thecapture probe, in which the hybridization complex is attached to asupport via the immobilized probe, and separating the hybridizationcomplex attached to the support from other sample components. In apreferred embodiment, the denaturant chemical is 8 M urea and the firstincubation is at about 95° C. for about 10 minutes or less. In apreferred embodiment, the denaturant chemical is imidazole at aconcentration from 0.5 M to 4.2 M, and the first incubation is at about60° C. for about 1 to 15 minutes. Other preferred embodiments, useimidazole at a concentration from 3.0 M to 3.5 M, and incubate at afirst temperature of 60° C. for about 1 to 15 minutes. In otherpreferred embodiments, imidazole at a concentration from 2.0 M to 2.7 Mand the first incubation is at about 90° C. to 95° C. for about 3 to 10minutes. In some preferred embodiments, the denaturant chemical isimidazole at a concentration of 2.7 M, the first incubating temperatureis about 75° C. to 95° C. for about 3 to 15 minutes, and the methodfurther includes incubating the reaction mixture at about 60° C. forabout 20 minutes between the first and second incubating steps. In apreferred embodiment that uses imidazole at a concentration of 2.7 M,the first incubating temperature is about 95° C. for about 3 to 15minutes, and the method includes incubating the reaction mixture atabout 60° C. for about 20 minutes between the first and secondincubating steps. In some embodiments, the target nucleic acid is acompletely or partially double-stranded nucleic acid, or a nucleic acidthat includes other secondary or tertiary structure. In one embodiment,the capture probe is made up of a target-specific sequence that binds tothe target nucleic acid and a tail region that binds to the immobilizedprobe via a specific binding partner. In a preferred embodiment, thecapture probe's tail region binds to the immobilized probe byhybridizing specifically to a complementary sequence of the immobilizedprobe. Some preferred embodiments also include detecting the targetnucleic acid or an in vitro amplification product made from the targetnucleic acid after separating the hybridization complex attached to thesupport from other sample components.

A method is disclosed for isolating a target nucleic acid of interestfrom a sample that includes the steps of mixing a sample containing atarget nucleic acid with a capture probe that hybridizes specifically toa target sequence in the target nucleic acid in a solution phase thatcontains a denaturant chemical and an immobilized probe that bindsspecifically to the capture probe, to provide a reaction mixture,incubating the reaction mixture at about 25° C. for about 1 to 60minutes, thereby forming a hybridization complex made up of the captureprobe hybridized specifically to the target nucleic acid and theimmobilized probe bound specifically to the capture probe, in which thehybridization complex is attached to a support via the immobilizedprobe, and separating the hybridization complex attached to the supportfrom other sample components. In a preferred embodiment, the denaturantchemical is urea at a concentration of about 1 M. In other preferredembodiments, the denaturant chemical is imidazole at a concentrationbetween 0.05 M and 0.5 M and incubating is for about 2 to 30 minutes. Ina preferred embodiment, imidazole is at a concentration of about 0.5 Mand incubating is for about 15 minutes.

A composition for specific capture of a target nucleic acid is disclosedthat includes at least one target nucleic acid, at least one captureprobe that hybridizes specifically to a target sequence in the targetnucleic acid, an immobilized probe that binds specifically to thecapture probe, and a solution phase hybridization mixture that containsimidazole at a concentration from 0.05 M to 4.2 M or urea at aconcentration from 1 to 8 M. In some preferred embodiments, the mixturecontains from 0.05 to 0.5 M imidazole, whereas in other preferredembodiments, the mixture contains from 1.7 M to 3.5 M imidazole. In somepreferred embodiments, the hybridization mixture contains from 2.0 M to2.7 M imidazole. In a preferred embodiment, the composition includes afirst capture probe that hybridizes specifically to a first targetsequence and a second capture probe that hybridizes specifically to asecond target sequence which is different from the first targetsequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing results of target capture in the presenceof 2.7 M imidazole in mixtures incubated at room temperature, 64° C.,85° C., and 95° C. for three nucleic acid targets, HBV subtype A (mediumshaded bars), HBV subtype B (dark shaded bars), and HBV subtype C (lightshaded bars), compared to target capture performed at room temperaturein mixtures without imidazole.

FIG. 2 is a bar graph showing results of target capture of HBV subtype Bnucleic acid from mixtures containing 2.7 M imidazole incubated for 1,3, 5, and 7 min at 95° C. compared to results of target captureperformed in mixtures without imidazole.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed methods of target capture isolate specific target nucleicacids from a sample by using hybridization of a capture probe to thetarget nucleic acid in a mixture using hybridization conditions thatinclude a denaturant chemical in a solution, such as imidazole or urea,and incubation of the mixture in a temperature range of about 60° C. to95° C. for about 2 to 15 minutes. In preferred embodiments, the targetnucleic acid is completely or partially double-stranded DNA. Preferredembodiments of the method use hybridization conditions that include 1.7Mto 3.2 M imidazole in a solution that contains a capture probe and itstarget that is incubated at 75° C. to 95° C. for about 3 to 7 minutes.

Another disclosed method isolates a target nucleic acid of interest froma sample in a single incubation step by using specific hybridization ofa capture probe to the target nucleic acid and binding of the captureprobe to an immobilized probe in a mixture that includes a denaturantchemical in solution, such as 0.5 M imidazole or 1 M urea, incubated atroom temperature (about 25° C.) for about 15 to 60 minutes. In preferredembodiments, the capture probe is a nucleic acid oligomer made up of2′-methoxy RNA groups or includes one or more LNA residues. In apreferred embodiment, 30 minutes of room temperature incubation is usedto capture the target nucleic acid of interest using a target-specificcapture probe.

These methods are particularly useful for isolating a nucleic acid ofinterest that is completely or partially double-stranded, or containsother secondary or tertiary structure. Some embodiments use asynergistic effect on hybridization achieved by using a denaturantchemical in solution in a mixture that includes the capture probe andtarget nucleic acid that is incubated at about 60° C. to 95° C. for arelatively short time, e.g., about 1 to 15 minutes. Preferredembodiments of the target capture method use hybridization conditions inwhich the denaturant chemical is imidazole at a concentration in a rangeof about 2.1 M to 4.2 M. Other embodiments use hybridization conditionsin which imidazole is in a concentration range of about 2.7 M to 3.2 M.A preferred embodiment of the target capture method uses a hybridizationmixture that includes about 2.7 M imidazole in solution and at least onetarget-specific capture probe specific for at least one target nucleicacid to efficiently bind the target nucleic acid when hybridizationconditions include incubation of the mixture for 3 to 7 min at about 75°C. to 95° C., or more preferably at about 85° C. to 95° C.

Target capture methods described herein may be used with two or morecapture probes to capture the same target nucleic acid from a sample orto capture two or more different target nucleic acids from the samesample, using one set of target capture conditions. That is, two or moredifferent target-specific capture probes may act in the same reactionmixture and same conditions, each probe specific for its intended targetsequence, so long as all the probes exhibit substantially similarhybridization characteristics in the target capture conditions used. Forexample, one embodiment is a target capture method that uses a firstcapture probe specific for a first target nucleic acid and a secondcapture probe specific for a second target nucleic acid that isdifferent from the first target nucleic acid, where the first and secondcapture probes exhibit substantially similar hybridizationcharacteristics for their respective targets in a single reactionmixture that includes imidazole and is incubated at about 60° C. to 95°C. for a short time before separation of the captured first and secondtarget nucleic acids from other sample components. Another embodiment isa target capture method that uses a first capture probe specific for afirst sequence in a target nucleic acid and a second capture probespecific for a second sequence in the same target nucleic acid, wherethe first and second capture probes exhibit substantially similarhybridization characteristics for their respective target sequences in asingle reaction mixture that includes imidazole and is incubated atabout 60° C. to 95° C. for a short time before separation of thecaptured target nucleic acid from other sample components.

Compositions disclosed herein include a target-specific capture probe ina hybridization reaction mixture that includes a denaturant chemical,such as urea or imidazole, to increase efficiency of specifichybridization of the capture probe oligomer to its target sequence,particularly when the target sequence is in a nucleic acid that ispartially or fully double stranded, or contains other secondary ortertiary structure. Compositions include components for making ahybridization reaction mixture that includes a denaturant chemical,preferably imidazole, in a solution that may contain one or more targetcapture reaction components, such as at least one capture probe specificfor the intended target nucleic acid, an immobilized binding partnerthat binds to the capture probe, or chemical components in ahybridization solution (e.g., salts, buffering agents). Thesecompositions include kits for performing specific polynucleotide targetcapture that include at least one capture probe specific for an intendedtarget nucleic acid and a denaturant chemical, preferably imidazole, ina solution phase mixture. Preferred kit embodiments contain a solutionthat contains imidazole, a capture probe oligomer specific for theintended target nucleic acid, and an immobilized binding partner for thecapture probe. Other kit embodiments also include one or more componentsused in treating the isolated captured target nucleic acids in an assaythat detects the target nucleic acid in a sample, such as a washingsolution for purifying the captured target nucleic acid from othersample components, or components used in in vitro amplification of asequence contained in the captured target nucleic acid, and/orcomponents used in detection of the captured target nucleic acid oramplification products made from the captured target nucleic acid.

Preferred target capture reagents include at least one capture probethat hybridizes specifically to a sequence in the nucleic acid ofinterest (i.e., target nucleic acid) and sufficient denaturant chemicalto make a hybridization mixture when mixed with a sample containing thetarget nucleic acid to produce the synergistic effect when the mixtureis incubated for a short time at temperatures in a range of about 60° C.to 95° C. Such a mixture may be produced, e.g., by combining apredetermined amount of a target capture reagent containing the captureprobe with a sample containing the target nucleic acid. The mixture forachieving these hybridization conditions may be made by mixing thedenaturant chemical with the sample containing the target nucleic acidsimultaneously with introduction of the capture probe, or the denaturantchemical may be added before or after the capture probe is mixed withthe sample. Preferred embodiments use a minimum of addition steps tomake the final mixture used in the hybridization conditions for targetcapture. A preferred target capture reagent includes both thetarget-specific capture probe and an immobilized binding partner thatbinds to the capture probe to separated the capture probe-target nucleicacid complex efficiently from other sample components. It will beappreciated that the reagents may include one or more target-specificcapture probes, e.g., two or more target-specific capture probes, solong as the probes have substantially the same hybridizationcharacteristics to produce efficient target capture for their respectiveintended target sequences in the same hybridization conditions thatinclude the denaturant chemical and incubation temperatures chosen. Onepreferred reagent embodiment includes a first capture probe specific fora first target nucleic acid and a second capture probe specific for asecond target nucleic acid that is different from the first targetnucleic acid, where the first and second capture probes exhibitsubstantially similar hybridization kinetics for their respectivetargets in a single hybridization condition that includes imidazole andincubation of the hybridization mixture from between 25° C. to 95° C.for about 30 minutes or less. Another reagent embodiment includes afirst capture probe specific for a first sequence in a target nucleicacid and a second capture probe specific for a second sequence in thesame target nucleic acid, where the first and second capture probesexhibit substantially similar hybridization kinetics for theirrespective target sequences in a single hybridization condition used intarget capture.

Compositions and methods described herein are particularly useful forisolation of target nucleic acids that are partially or completelydouble stranded (e.g., dsDNA) or contain secondary structure (e.g.,hairpin structures) under relatively mild conditions. Other knownisolation methods often include a step of denaturing the target nucleicacid (e.g., boiling for 5-10 min) to make the target nucleic acid singlestranded, but such treatments are difficult to perform, may causecontamination of laboratory personnel or equipment if a container opensor explodes, and may produce structures that make nucleic acid isolationinefficient, e.g., coagulation aggregates or damaged nucleic acids.Moreover, insufficient denaturation or reannealing of the denaturednucleic acids before target capture may result in suboptimal capture.

Methods and compositions described herein are useful for purifyingdesired nucleic acid sequences from a complex mixture, such as from asample that contains nucleic acids or cells, which may be treated byusing conventional methods to release intracellular nucleic acids into asolution. The methods are useful for preparing nucleic acids for use inmolecular biology assays or procedures, such as diagnostic assays thatdetect a specific sequence, forensic tests that detect the presence ofbiological material, or tests to detect contaminants in water,environmental or food samples. Methods and compositions described hereinare useful for preparing nucleic acids for in vitro nucleic acidamplification which is used in many applications. Because the methodsconcentrate the target nucleic acids and remove them from other samplecomponents that might interfere with subsequent assay steps, thesemethods are useful for improving assay specificity and/or sensitivity.The methods are relatively simple to perform, making them useful forscreening specimens manually or using automation.

A “sample” or “specimen” refers to any composition in which a targetnucleic acid may exist as part of a mixture of components, e.g., inwater or environmental samples, food stuffs, materials collected forforensic analysis, or biopsy samples for diagnostic testing. “Biologicalsample” refers to any tissue or material derived from a living or deadorganism which may contain a target nucleic acid, including, e.g.,cells, tissues, lysates made from cells or tissues, sputum, peripheralblood, plasma, serum, cervical swab samples, biopsy tissues (e.g., lymphnodes), respiratory tissue or exudates, gastrointestinal tissue, urine,feces, semen, or other fluids or materials. A sample may be treated tophysically disrupt tissue and/or cell structure to release intracellularcomponents into a solution which may contain enzymes, buffers, salts,detergents and other compounds, such as are used to prepare a sample foranalysis by using standard methods.

“Nucleic acid” refers to a multimeric compound comprising nucleotides oranalogs which have nitrogenous heterocyclic bases or base analogs linkedtogether to form a polynucleotide, including conventional RNA, DNA,mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds(“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioatelinkages, methylphosphonate linkages, or combinations thereof. Sugarmoieties of a nucleic acid may be ribose, deoxyribose, or similarcompounds with substitutions, e.g., 2′ methoxy or 2′ halidesubstitutions. Nitrogenous bases may be conventional bases (A, G, C, T,U), analogs thereof (e.g., inosine or others; see The Biochemistry ofthe Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992),derivatives of purines or pyrimidines (e.g., N⁴-methyl deoxygaunosine,deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases withsubstituent groups at the 5 or 6 position, purine bases with asubstituent at the 2, 6 or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines; U.S. Pat. No.5,378,825 and PCT No. WO 93/13121). Nucleic acids may include one ormore “abasic” residues where the backbone includes no nitrogenous basefor one or more positions (U.S. Pat. No. 5,585,481). A nucleic acid mayinclude only conventional RNA or DNA sugars, bases and linkages, or mayinclude both conventional components and substitutions (e.g.,conventional bases with 2′ methoxy linkages, or polymers containing bothconventional bases and analogs). The term includes “locked nucleic acid”(LNA), an analogue containing one or more LNA nucleotide monomers with abicyclic furanose unit locked in an RNA mimicking sugar conformation,which enhance hybridization affinity for complementary RNA and DNAsequences (Vester et al., 2004, Biochemistry 43(42):13233-41).Embodiments of oligomers that may affect stability of a hybridizationcomplex include PNA oligomers, oligomers that include 2′-methoxy or2′-fluoro substituted RNA, or oligomers that affect the overall charge,charge density, or steric associations of a hybridization complex,including oligomers that contain charged linkages (e.g.,phosphorothioates) or neutral groups (e.g., methylphosphonates).

“Oligomer” or “oligonucleotide” refers to a nucleic acid of generallyless than 1,000 nucleotides (nt), including those in a size range havinga lower limit of about 2 to 5 nt and an upper limit of about 500 to 900nt. Some preferred embodiments are oligomers in a size range with alower limit of about 5 to 15 nt and an upper limit of about 50 to 600nt, and other preferred embodiments are in a size range with a lowerlimit of about 10 to 20 nt and an upper limit of about 22 to 100 nt.Oligomers may be purified from naturally occurring sources, butpreferably are synthesized by using any well known enzymatic or chemicalmethod. Oligomers may be referred to by functional names (e.g., captureprobe, primer or promoter primer) which are understood to refer tooligomers.

“Capture probe”, “capture oligonucleotide”, or “capture oligomer” refersto a nucleic acid oligomer that specifically hybridizes to a targetsequence in a target nucleic acid by base pairing and joins to a bindingpartner on an immobilized probe to capture the target nucleic acid to asupport. A preferred embodiment of a capture oligomer includes twobinding regions: a target-specific region and an immobilizedprobe-binding region, usually on the same oligomer, although the tworegions may be present on two different oligomers joined together by oneor more linkers.

“Immobilized probe”, “immobilized oligomer” or “immobilized nucleicacid” refers to a nucleic acid binding partner that joins a captureoligomer to a support, directly or indirectly. An immobilized probejoined to a support facilitates separation of a capture probe boundtarget from unbound material in a sample. Any support may be used (e.g.,matrices or particles in solution), which may be made of any of avariety of materials (e.g., nylon, nitrocellulose, glass, polyacrylate,mixed polymers, polystyrene, silane polypropylene, or metal). Preferredsupports are magnetically attractable particles, e.g., monodisperseparamagnetic beads (uniform size ±5%) to which an immobilized probe isjoined directly (e.g., via covalent linkage, chelation, or ionicinteraction) or indirectly (e.g., via a linker), where the joining isstable during nucleic acid hybridization conditions.

“Separating” or “purifying” refers to removing one or more components ofa sample from one or more other sample components, e.g., removing somenucleic acids from a generally aqueous solution that may also containproteins, carbohydrates, lipids, or other nucleic acids. In preferredembodiments, a separating or purifying step removes the target nucleicacid from at least about 70%, more preferably at least about 90% and,even more preferably, at least about 95% of the other sample components.

“Hybridization conditions” refer to the cumulative physical and chemicalconditions under which nucleic acid sequences that are completely orpartially complementary form a hybridization duplex or complex, usuallyby standard base pairing. Such conditions are well known to thoseskilled in the art, are predictable based on sequence composition of thenucleic acids involved in hybridization complex formation, or may bedetermined empirically by using routine testing (e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) at §§1.90-1.91,7.37-7.57, 9.47-9.51, and 11.47-11.57, particularly §§9.50-9.51,11.12-11.13, 11.45-11.47 and 11.55-11.57).

“Sufficiently complementary” means that a contiguous nucleic acid basesequence is capable of hybridizing to another base sequence by standardbase pairing (hydrogen bonding) between a series of complementary bases.Complementary sequences may be completely complementary at each positionin an oligomer sequence relative to its target sequence by usingstandard base pairing (e.g., G:C, A:T or A:U pairing) or sequences maycontain one or more positions that are not complementary by base pairing(including abasic residues), but such sequences are sufficientlycomplementary because the entire oligomer sequence is capable ofspecifically hybridizing with its target sequence in appropriatehybridization conditions. Contiguous bases in an oligomer are at least80%, preferably at least 90%, and more preferably completelycomplementary to the intended target sequence.

“Nucleic acid amplification” refers to any well known in vitro procedurethat produces multiple copies of a target nucleic acid sequence, or itscomplementary sequence, or fragments thereof (i.e., an amplifiedsequence containing less than the complete target nucleic acid).Examples of well known procedures include transcription associatedmethods, such as transcription-mediated amplification (TMA), nucleicacid sequence-based amplification (NASBA) and others (U.S. Pat. Nos.5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246),replicase-mediated amplification (U.S. Pat. No. 4,786,600), thepolymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195, 4,683,202,and 4,800,159), ligase chain reaction (LCR) (EP Pat. App. 0320308) andstrand-displacement amplification (SDA) (U.S. Pat. No. 5,422,252).

“Detection probe” refers to a nucleic acid oligomer that hybridizesspecifically to a target nucleic acid sequence, including an amplifiedsequence, under conditions that promote hybridization, to allowdetection of the target nucleic acid. Detection may either be direct(i.e., a probe hybridized directly to the target) or indirect (i.e., aprobe hybridized to an intermediate structure that links the probe tothe target). A probe's target sequence generally refers to the specificsequence within a larger sequence which the probe hybridizesspecifically. A detection probe may include target-specific sequencesand other sequences or structures that contribute to the probe'sthree-dimensional structure, depending on whether the target sequence ispresent (U.S. Pat. Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412).

“Label” refers to a moiety or compound that is detected or leads to adetectable signal, which may be joined directly or indirectly to anucleic acid probe. Embodiments that use direct joining include use ofcovalent bonds or non-covalent interactions, e.g., hydrogen bonding,hydrophobic or ionic interactions, and chelate or coordination complexformation. Embodiments that use indirect joining include use of abridging moiety or linker, e.g., via an antibody or additionaloligonucleotide(s), which may be used to amplify a detectable signal.Any detectable moiety may be a label, e.g., radionuclide, ligand such asbiotin or avidin, enzyme, enzyme substrate, reactive group, chromophoresuch as a dye or particle (e.g., latex or metal bead) that imparts adetectable color, luminescent compound (e.g. bioluminescent,phosphorescent or chemiluminescent compound), and fluorescent compound.Preferred embodiments include a “homogeneous detectable label” that isdetectable in a homogeneous assay system in which, in a mixture, boundlabeled probe exhibits a detectable change compared to unbound labeledprobe, which allows the label to be detected in a homogeneous fashionwithout physically removing hybridized from unhybridized labeled probe(U.S. Pat. Nos. 5,283,174, 5,656,207 and 5,658,737). Preferredhomogeneous detectable labels include chemiluminescent compounds, morepreferably acridinium ester (“AE”) compounds, such as standard AE or AEderivatives which are well known (U.S. Pat. Nos. 5,656,207, 5,658,737,and 5,639,604). Methods of synthesizing labels, attaching labels tonucleic acids, and detecting signals from labels are well known (e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Chapt.10, and U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and4,581,333).

Unless defined otherwise, technical terms used herein have the samemeaning as commonly understood by those skilled in the art or indefinitions found in technical literature, e.g., Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), The Harper Collins Dictionary ofBiology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), andsimilar publications. Unless described otherwise, techniques employed orcontemplated herein are standard well known methods.

The disclosed target capture methods may result from increasing theefficiency of hybridization between a nucleic acid probe and a targetnucleic acid in a solution that includes other components. These methodsmay use a synergistic effect that results when a hybridization mixturecontaining a denaturant chemical, e.g., imidazole or urea, and isincubated for a short time at elevated temperature, e.g., 60° C. to 95°C. before separation of a hybridization complex that includes thecapture probe and target nucleic acid from other mixture components.Preferred embodiments use incubation temperatures of about 75° C. to 95°C. and imidazole in the hybridization mixture to increase the efficiencyand rate of specific hybridization between the capture probe and itstarget sequence. Other preferred embodiments are compositions made up ofa solution that contains at least one capture probe oligomer and itsintended target nucleic acid in a solution that includes about 1.7 M to2.7 M imidazole.

Another target capture method, referred to as a “standard” method, usessimilar steps but does not include a denaturant chemical in the mixture(U.S. Pat. Nos. 6,110,678, 6,280,952, and 6,534,273). Methods disclosedherein provide efficient target capture under relatively mildconditions, particularly for partially or completely double-strandedtarget nucleic acids (e.g., dsDNA, dsRNA or DNA/RNA hybrids). Thedisclosed methods are useful for improving assay performance andsensitivity, particularly for assays that use the isolated targetnucleic acids in subsequent procedures, such as in vitro amplificationand/or detection. The relatively mild conditions described hereininclude a denaturant chemical in a hybridization mixture which may beused at room temperature or heated for a short time, e.g., about 60-95°C. for 15 minutes or less.

Preferred capture probe oligomers include a target-specific sequencethat binds specifically to a sequence in the target nucleic acid, and amoiety that binds to an immobilized probe for separation of thehybridization complex that includes the target nucleic acid from therest of the mixture. Some preferred embodiments of capture oligomersinclude a tail sequence (e.g., a substantially homopolymeric sequence)that hybridizes to a complementary immobilized sequence on a support.Other capture probe embodiments bind to an immobilized probe by using amoiety that is a member of a binding pair (e.g., biotinylated DNA andimmobilized avidin or streptavidin). In an embodiment that uses acapture probe with a target-specific sequence and a tail sequence, thecapture probe is mixed with a sample that contains the target nucleicacid and, under hybridizing conditions that include the denaturantchemical, the capture probe's target-specific portion hybridizesspecifically to its target sequence and the tail portion hybridizes to acomplementary immobilized sequence to allow the target sequence linkedto the support to be separated from other components. In preferredembodiments, the target-specific portion hybridizes to the targetnucleic acid in a first step and the tail portion hybridizes to theimmobilized sequence in a second step, which makes use of favorablesolution phase hybridization kinetics in the first step. In preferredembodiments, hybridization conditions include a soluble denaturantchemical in the mixture that contains the capture probe and targetnucleic acid and incubation of the mixture at 60° C. to 95° C.,preferably 75° C. to 95° C., for about 15 min or less to permitformation of a hybridization duplex of the capture probe and targetnucleic acid, followed by incubation at a lower temperature (e.g., about25° C. to 42° C.) to permit formation of a hybridization complex made upof the target nucleic acid, capture oligomer, and immobilized probe. Inembodiments in which the capture probe and immobilized probe bind vianon-nucleic acid binding pair members, hybridization conditions allowformation of the capture probe-target hybridization duplex and bindingof the capture probe and immobilized probe via the binding pair members.

An immobilized probe may be connected to a support by any linkage thatis stable in the hybridization conditions used in the target capturemethod. Preferred embodiments use a support of monodisperse particleswhich can be retrieved from solution by using known methods, e.g.,centrifugation, filtration, magnetic attraction, or other physical orelectrochemical separation. The support with the attached hybridizationcomplex that includes the capture probe and target nucleic acid isseparated from other sample components. In some embodiments,hybridization complexes bound to the support are washed one or moretimes under conditions that maintain complexes on the support to furtherseparate other components, including other nucleic acids, from thecaptured target nucleic acid. The target nucleic acid is isolated andconcentrated on the support, i.e., the target nucleic acid concentrationon the support that is higher than in the initial sample. The isolatedtarget nucleic acid, attached to or eluted from the support, may be usedin a variety of subsequent processes such as in vitro amplificationand/or detection.

Target capture methods described herein may be used to isolate two ormore target nucleic acids from the same sample simultaneously byincluding two or more capture probes in the hybridization mixture, eachcapture probe specific for a target sequence. For example, ahybridization mixture may include a first capture probe specific for afirst target and a second capture probe specific for a second target,where each capture probe hybridizes to its intended target under thesame hybridization conditions. Each capture probe may bind to the sameimmobilized probe or may bind to an immobilized probe specific for theindividual capture probe. In one embodiment, the first and secondcapture probes both contain a poly-A tail region and bind to the sameimmobilized probe that includes a complementary poly-T sequence, thuspurifying the first and second targets on the same support. In anembodiment, the first capture probe binds to a first immobilized probeon a first support, and the second capture probe binds to a secondimmobilized probe on a second support, thus purifying the first targeton the first support and the second target on the second support. Whenthe first and second supports exhibit different separationcharacteristics, the first target on the first support is readilyseparated from the second target on the second support, even though allof capture complexes formed in the same reaction mixture. These methodsmay be used to isolate many targets from a single sample by usingcombinations of different capture probes which all function insubstantially the same hybridization conditions to isolate many targetsfrom a mixture.

Initial target capture tests were performed by using samples thatcontained a known amount of a target RNA (Chlamydia trachomatis 23SrRNA) which was hybridized to a labeled detection probe to make alabeled target complex. Target capture was performed on the labeledtarget complex by using a capture probe that contains a 5′target-specific sequence complementary to a sequence contained in therRNA and a 3′ tail sequence complementary to oligonucleotidesimmobilized on magnetic beads. Target capture mixtures were incubatedunder different hybridization conditions, including in solutions withand without a denaturant chemical (urea or imidazole), at roomtemperature to 60° C., for various times from 1 to 60 min. The capturedlabeled target complex on the beads was separated from the other mixturecomponents and the target was detected by measuring a signal from theattached labeled probe. Many capture probes specific for differenttarget sequences in the rRNA were tested for target capture efficiencyat room temperature and most showed increased capture efficiency whenimidazole was included (e.g., 0.05 M to 1 M). Some of the capture probesthat were efficient at target capture at room temperature weresubsequently tested in different conditions to determine hybridizationconditions that increased efficiency and/or kinetics of target capture.For example, some target capture tests also included one or more helperoligonucleotides complementary to a sequence in the rRNA to facilitatebinding of another complementary sequence to the target RNA (U.S. Pat.No. 5,030,557). Generally, capture probes were synthesized with 2′methoxy RNA in the target-specific portion and DNA in the tail portion.Relative efficiencies of the target capture conditions were determinedby measuring the signal produced from detection probes bound to thecaptured target after it was separated from the mixture. Differentcapture probes performed at different efficiencies under the sameconditions, but almost all of the tested capture probes showed anincrease in target capture efficiency when imidazole was included in thereaction mixture. For example, efficient capture of the rRNA target (75%to 90%) with relatively fast kinetics was observed when target capturemixtures contained 0.5 M imidazole and were incubated at 60° C. for 1minute, 42° C. for 10 minutes, or room temperature for 15 minutes. Theseexperiments demonstrated that hybridization conditions that included adenaturant chemical generally increased the efficiency and kinetics oftarget capture compared to similar hybridization conditions that did notinclude a denaturant chemical in the reaction mixture. Many of thesetests showed higher background signals (e.g., in controls that did notcontain the target capture probe) when imidazole was present compared tosimilar assays performed without imidazole, but this was substantiallyeliminated in later tests by modifying the detection conditions (i.e.,increasing the pH of the selection reagent to pH 9.2 and incubating theselection step longer, e.g., 5-10 min). Assays performed with imidazolein the target capture reaction incubated at lower temperatures (25° C.to 42° C.) typically resulted in less total detectable signal comparedto target capture assays performed using the same capture probe in areaction mixture without imidazole incubated at higher temperature (60°C.). An increase in target capture efficiency, however, was repeatedlyobserved when reaction mixtures contained imidazole compared to matchedreactions that did not include imidazole in the mixture, for manydifferent probes and incubation conditions that were tested.

Embodiments of efficient target capture methods were demonstrated in amodel system that used a synthetic partially dsDNA target that wascaptured by using a capture probe that included a target-specificsequence and a tail portion which was complementary to an immobilizedoligomer on a particulate support. These components were mixed in asolution containing salts and buffering agents with differentconcentrations of denaturant chemical and incubated for a short time (10min or less) at different temperatures (e.g., about 60° C. to 95° C.)for hybridization of the target-specific portion of the capture probewith its target nucleic acid, and then at a lower temperature (e.g., RTto 42° C.) for hybridization of the tail portion to the immobilizedoligomer. Particles with the attached complexes were separated from theother components in the mixture and the captured target nucleic acid wasdetected by detecting a signal from a labeled detection probe hybridizedto the target nucleic acid or an amplification product made from thecaptured target, measured in a homogeneous assay. The model systemdemonstrated the unexpected result that hybridization between thetarget-specific region of the capture probe and its target sequence wasefficient under relatively mild incubation conditions when a denaturantchemical was included in the hybridization mixture, but the denaturantchemical did not interfere with hybridization between the capture probetail and the immobilized probe, resulting in an efficient capture of thetarget. Although not wishing to be bound to a particular theory ormechanism, this may result from increased denaturation of thedouble-stranded portion of the target DNA, thus making the targetsequence accessible to hybridization with the capture probe while notinhibiting other target capture steps.

Viral targets were also used to demonstrate increased target captureefficiency by using the compositions and methods of target capturedisclosed herein. One target was BK virus (BKV) which contains a fullydsDNA genome and another target was hepatitis B virus (HBV), which has apartially or fully double-stranded genome depending on its replicationphase. When these target viruses were tested using a standard targetcapture procedure that did not include a denaturant chemical (describedin U.S. Pat. Nos. 6,110,678, 6,280,952, and 6,534,273), capture of theviral genomes was suboptimal, e.g, retrieving less than 60% of thetarget nucleic acid in the sample. Assays for the viruses that used thestandard target capture method were relatively insensitive even when thecaptured viral nucleic acids were amplified in vitro and amplifiedsequences were detected. Even when the sample containing viral DNA washeated at a high temperature (95° C.) to denature the target DNA beforeusing the standard target capture process, assay sensitivity showed onlymarginal improvement. In contrast, when embodiments of the efficienttarget capture method disclosed herein that included imidazole in thetarget capture reactions were used, the assay sensitivity improved.Embodiments of the efficient target capture process included acombination of including a denaturant chemical, imidazole or urea, inthe reaction mixture and heating the mixture at an initial phase in thetarget capture process, which resulted in a surprising synergisticeffect that greatly improved target capture efficiency and assaysensitivity. For example, one embodiment that included imidazole in thetarget capture process before in vitro nucleic acid amplificationresulted in 95% detection rates for HBV in samples that contained HBVsubtypes B, C and A (28-fold, 4-fold and 2-fold increased detectionrates, respectively, compared to assays that did not include theefficient target capture method). Another embodiment for used urea inthe target capture mixture which was incubated at 95° C. during aninitial step of the target capture process, after which the captured HBVDNA was amplified in vitro and amplified sequences were detected.Another embodiment for BKV DNA capture included imidazole in the targetcapture mixture that was incubated for a short time at high temperaturefollowed by a lower temperature, and separation of the captured BKV DNAfrom other components, which improved the assay sensitivity 10-foldcompared to a similar assay that used a standard target capture method.In the comparative assays, the captured BKV DNA was amplified in vitroand amplified BKV sequences were detected by using a labeled detectionprobe.

Examples are included to describe embodiments of the disclosed targetcapture methods and compositions. In some cases, after target capture,the captured nucleic acids were subjected to additional steps, e.g., invitro amplification and/or detection using a labeled probe, by usingknown methods (e.g., U.S. Pat. Nos. 5,399,491 and 5,554,516, foramplification; U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737, fordetection probe labeling, hybridization and detection steps). Someexamples describe assays performed with different embodiments of atarget capture process that includes a denaturant chemical, which may becompared to assays performed with a standard target capture process thatdoes not include a denaturant chemical (U.S. Pat. Nos. 6,110,678,6,280,952, and 6,534,273). Unless otherwise specified, reagents commonlyused in assays described below are as follows. Sample transport reagent:110 mM lithium lauryl sulfate (LLS), 15 mM NaH₂PO₄, 15 mM Na₂HPO₄, 1 mMEDTA, 1 mM EGTA, pH 6.7. Target Capture Reagent (TCR): 789 mM HEPES, 230mM succinic acid, 10% w/v LLS, 679 mM LiOH, 0.03% anti-foaming agent, pH6.4, and 100 μg/ml of paramagnetic particles (0.7-1.05 μparticles,SERA-MAG™ MG-CM, Seradyne, Inc., Indianapolis, Ind.) with (dT)₁₄oligomers covalently bound thereto, or (C-type) 250 mM HEPES, 1.88 MLiCl, 310 mM LiOH, 100 mM EDTA, pH 6.4, and 250 μg/ml of paramagneticparticles (0.7-1.05 μparticles, Sera-Mag™ MG-CM) with (dT)₁₄ oligomerscovalently bound thereto. Wash Solution: 10 mM HEPES, 150 mM NaCl, 6.5mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methylparaben, 0.01%(w/v) propylparaben, and 0.1% (w/v) sodium lauryl sulfate, pH 7.5.Amplification reagent: a concentrated solution mixed with other TMAreaction components to produce a mixture containing 47.6 mM Na-HEPES,12.5 mM N-acetyl-L-cysteine, 2.5% TRITON™ X-100, 54.8 mM KCl, 23 mMMgCl₂, 3 mM NaOH, 0.35 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 7.06 mMrATP, 1.35 mM rCTP, 1.35 mM UTP, 8.85 mM rGTP, 0.26 mM Na₂EDTA, 5% v/vglycerol, 2.9% trehalose, 0.225% ethanol, 0.075% methylparaben, 0.015%propylparaben, and 0.002% Phenol Red, pH 7.5-7.6. Primers and/or probesmay be in the amplification reagent or added separately to a mixture.Enzymes for TMA: about 90 U/μl of MMLV reverse transcriptase (RT) andabout 20 U/μl of T7 RNA polymerase per reaction (1 U of RT incorporates1 nmol of dTTP in 10 min at 37° C. using 200-400 μM oligo dT-primedpolyA template, and 1 U of T7 RNA polymerase incorporates 1 nmol of ATPinto RNA in 1 hr at 37° C. using a T7 promoter in a DNA template). ProbeReagent: AE-labeled detection probes in a solution of (a) 100 mMLi-succinate, 3% (w/v) LLS, 10 mM mercaptoethanesulfonate (MES), and 3%(w/v) polyvinylpyrrolidon, or (b) 100 mM Li-succinate, 0.1% (w/v) LLS,and 10 mM MES. Hybridization Reagent: (C-type) 100 mM succinic acid, 2%(w/v) LLS, 100 mM LiOH, 15 mM aldrithiol-2, 1.2 M LiCl, 20 mM EDTA, and3.0% (v/v) ethanol, pH 4.7; or (P-type) 190 mM succinic acid, 17% (w/v)LLS, 3 mM EDTA, and 3 mM EGTA, pH 5.1. Selection Reagent: 600 mM boricacid, 182.5 mM NaOH, 1% (v/v) octoxynol (TRITON® X-100), pH 8.5 or pH9.2, to hydrolyze AE labels on unhybridized detection probe oligomers.Detection Reagents comprise Detect Reagent I: 1 mM nitric acid and 32 mMH₂O₂, and Detect Reagent II: 1.5 M NaOH, to produce chemiluminescencefrom AE labels (see U.S. Pat. Nos. 5,283,174, 5,656,744, and 5,658,737).

For comparison to the target capture methods that include a denaturantchemical, the standard target capture procedure is described briefly.The standard process typically mixes a sample that contains the targetnucleic acid (RNA or DNA) with about 1.75 pmols of a capture probespecific for the target nucleic acid (i.e., probe that hybridizesspecifically to a sequence contained in the target nucleic acid in thehybridization conditions used) and about 100 μg of immobilized probeattached to paramagnetic particles (dT₁₄ probes attached to 0.7-1.05μparticles (Seradyne) by using carbodiimide chemistry (Lund, et al.,1988, Nuc. Acids Res. 16:10861-10880)) in target capture reagent. Themixture may include an amplification oligomer (e.g., primer) thathybridizes to the target nucleic acid (U.S. Pat. No. 6,534,273). Thestandard target capture mixture is heated at 55° C. to 60° C. for about15 to 30 min, then cooled to room temperature (RT) for 5 to 15 min, toallow sequential hybridization of the capture probe and target nucleicacid and then the immobilized probe to the capture probe:target nucleicacid complex. A magnetic field is applied to separate particles withattached complexes from the solution phase and concentrate them in thecontainer (U.S. Pat. No. 4,895,650) and the supernatant is removed.Particles are washed by suspending them in Wash Solution (e.g., 1 ml atRT) and repeating the magnetic separation.

Captured target nucleic acids may be detected by using a detectionprocess or may be treated by in vitro nucleic acid amplification toamplify part of the target nucleic acid sequence which is detected. Forexample, for transcription mediated amplification (TMA), washedparticles are suspended in 75 μl of amplification reagent with primersand enzymes added to make a mixture that is incubated at 41.5-42° C. for1-2 hr (U.S. Pat. Nos. 5,399,491 and 5,554,516). Amplified sequences maybe detected by using an AE-labeled probe that hybridizes specifically toan amplified sequence and chemiluminescence from the AE label on boundprobes is detected, expressed as relative light units (RLU) (U.S. Pat.No. 5,658,737, see column 25, lines 27-46, Nelson et al., 1996, Biochem.35:8429-8438 at 8432), although any of a variety of in vitroamplification methods and/or detection methods may be used.

Target capture methods described herein include a denaturant chemical,preferably imidazole, in a solution phase mixture that includes acapture probe specific for the intended target nucleic acid, reagentsfor making a mixture for promoting nucleic acid hybridization (e.g.,salts, buffering agents), a target nucleic acid (generally added from asample), and an added immobilized probe, preferably in a suspension. Atarget capture mixture may include additional oligomers used in anassay, e.g., helper oligomers, in vitro amplification primer oligomers,and/or detection probe oligomers. In some embodiments, the denaturantchemical is in target capture reagents that include the capture probewhich are mixed with the sample containing the target nucleic acid tomake a mixture that is incubated at an elevated temperature (e.g., 60°C. to 95° C.) for a brief time (e.g., 1 to 15 min). In a preferredembodiment, the mixture is incubated at 75° C. to 95° C. for about 3min, and then the target capture process is performed. Although specifictemperatures and incubation times used in particular embodiments of theefficient target capture method may vary based on the chosen combinationof a particular capture probe and target nucleic acid, the methodincludes a denaturant chemical in the target capture mixture which isincubated for a short time (e.g., 1 to 30 min, preferably 1 to 15 min)at hybridization temperature (e.g., room temperature to 95° C.) beforethe separation step that removes the captured target from the mixture.In addition to increasing assay sensitivity, the target capture methodsdescribed herein are easy to perform, manually or in an automatedsystem, and add only a few minutes to the total assay time. Embodimentsof the efficient target capture method disclosed herein are useful forimproving assay sensitivity for target nucleic acids that are partiallyor completely double-stranded, or contain regions of secondary ortertiary structure.

Example 1 Target Capture of RNA Known to have Secondary and TertiaryStructure

This example demonstrates that hybridization conditions that includeimidazole generally increased efficiency and kinetics of target captureof a RNA known to have secondary and tertiary structure (23S rRNA) atrelatively low incubation temperatures, e.g., RT to about 42° C. formany capture probes. Secondary and/or tertiary structure in the targetRNA may inhibit interaction with a capture probe and limit targetcapture. Experiments were performed by using a known amount of Chlamydiatrachomatis 23S rRNA which was hybridized to a labeled detection probecomplementary to a sequence in the rRNA to label the target RNA beforeit was captured. Typically, 200 fmole of 23S rRNA was hybridized with 1pmole of an AE-labeled synthetic oligonucleotide in hybridizationreagent (60° C. for 30 min, followed by cooling to RT). An aliquot ofthe hybridized detection probe:target mixture was mixed with targetcapture reagent (TCR) containing a capture probe, immobilized probe,with or without a known concentration of denaturant chemical, andmixture was incubated at RT (about 25° C.) to 60° C. for 1 to 60 minbefore collection of the captured target. The capture probes (SEQ IDNos. 6 to 30) each contained a 5′ target-specific sequence complementaryto a sequence contained in the rRNA and a 3′ A₃₀ tail sequence,synthesized with 2′ methoxy RNA in the target-specific region and a DNAtail region. Immobilized probes were dT₁₅ oligonucleotides attached tomagnetic microparticles as supports (SERA-MAG™). Some assays included inthe target capture reaction mixture one or more helper oligonucleotidescomplementary to a separate sequence in the rRNA to facilitate bindingof another complementary sequence to the target rRNA (U.S. Pat. No.5,030,557, Hogan et al.). Denaturant chemicals were 1 M urea or 0.05 Mto 1.8 M imidazole. Following target capture, the labeled rRNA targetsattached to magnetic particles were separated from the solution phase byusing magnetic attraction, the supernatant was removed, particles withattached complexes were washed multiple times with a wash solution, andchemiluminescence from the labeled probe bound to the target, followinghydrolysis of AE labels in unhybridized detection probes using selectionreagent, was detected in a luminometer (as relative light units (RLU),substantially as described in U.S. Pat. No. 5,658,737).

Target capture assays performed at RT or 60° C. in the presence of 1Murea increased the detected RLU from the captured RNA (2.2 to 3.1-foldhigher) compared to signals detected from matched samples that weretreated identically except that no urea was in the target capturemixture. Because aqueous solutions of urea may decompose on heating(giving off NH₃), additional experiments were performed using imidazolein target capture mixtures.

In a series of assays, capture probes (SEQ ID Nos. 6 to 29) specific fordifferent target sequences in the 23S rRNA were tested individually fortarget capture efficiency at RT in the presence of imidazole (0.05-1 M)and all except one showed increased target capture when imidazole waspresent compared to a matched reactions in which the mixtures did notcontain imidazole (e.g., 1.3-fold to 8.8-fold increase in RLU detectedin the immobilized portion). Some of the capture probes (SEQ ID Nos. 6,8, 17, 20-22, 24 and 27) were tested subsequently by using differenthybridization conditions (incubated at 25° C., 42° C. and 60° C., withand without imidazole present). Relative efficiencies of target capturewere determined for each capture probe and condition by measuring thechemiluminescence produced from detection probes bound to the capturedtarget separated from the solution phase. The different capture probestested individually at RT in reactions that contained no imidazolecaptured the rRNA at different efficiencies (0% to 70% of initialtarget), whereas all of the capture probes except one captured more rRNAwhen 0.5 M imidazole was present in the reaction (2.6% to 89% of initialtarget). For example, one capture probe demonstrated high levels of rRNAcapture when reactions containing 0.5M imidazole were incubated at RT(about 75% of target captured by 15 min), or 42° C. (about 90% of targetcaptured by 10 min), or 60° C. (about 90% of target captured at 1 min).In contrast, in the absence of imidazole, at RT, target capture wasinefficient (11% of the target was captured by 60 min).

To measure the kinetics of target capture, capture probes (SEQ ID Nos.6, 20, 21 and 24) were used at RT in hybridization mixtures thatcontained no imidazole or 0.05 to 0.1 M imidazole to capture C.trachomatis 23S rRNA. The probes showed more target capture after 5 minincubation in reactions that contained 0.1 M imidazole compared tomatched reactions without imidazole. Capture probes (SEQ ID Nos. 6, 20,21) were tested in similar assays incubated at RT for 2, 15, and 30minutes. Increased target capture was seen for all of the probes in thepresence of 0.05 M imidazole after only 2 min incubation compared tomatched reactions without imidazole. One capture probe (SEQ ID NO:20)was tested subsequently in reactions that contained no or 0.05 Mimidazole, incubated at RT, 42° C., or 60° C., for 5, 15, or 30 minbefore measuring signal from the captured target. Target captureefficiency increased for all conditions that included imidazole in themixtures compared to the matched reactions without imidazole, with thegreatest increases seen after 5 min (2.2-fold increase at RT, 1.7-foldincreases at 42° C. and 60° C.) compared to longer incubations (1.7-foldincrease at RT, 1.3-fold increases at 42° C. and 60° C. for 15 min;1.4-fold increase at RT, 1.1 to 1.2-fold increases at 42° C. and 60° C.for 30 min). The results demonstrate increased kinetics of targetcapture in the presence of imidazole.

Example 2 Target Capture of a Synthetic Target Present in a PartiallyDouble-Stranded DNA

This example demonstrates efficiencies of target capture when thereaction mixtures contained different concentrations of imidazole, allincubated at 60° C. for 15 min, followed by incubation at RT for 30 min.For these tests, a partially double-stranded DNA (dsDNA) was made bysynthesizing a strand of SEQ ID NO:1 and its complementary strand (SEQID NO:2), hybridizing the two strands together (35 pmol of each in 20 μlof hybridization reagent (P-type), incubated at 60° C. for 1 hr, andcooled to RT). The resultant dsDNA contained, at one end, a 30 ntsingle-stranded DNA that is complementary to a detection probe (SEQ IDNO:4). Each target capture reaction mixture contained 10 fmoles of dsDNAtarget in 0.2 ml of sample transport reagent to which was added 0.2 mlof target capture reagent (TCR, C-type) containing 0 to 3.18 Mimidazole, 0.1 ml of TCR containing 50 μg of paramagnetic particles(Sera-Mag™) with covalently attached dT₁₄ oligomers, and 20 pmoles of acapture probe (SEQ ID NO:3) that contains a 5′ target-specific region(25 nt of LNA complementary to a sequence in the target strand of thedsDNA) and a 3′ polyA tail region. Reactions were incubated at 60° C.for 15 min, then at RT for 30 min, and the captured target DNA attachedto the magnetic supports was separated from the supernatant as describedabove. The pellet was washed once (using 0.5 ml of hybridizationreagent, P-type), and then AE labeled detection probe (100 fmoles of SEQID NO:4 in 0.1 ml of hybridization reagent, P-type) was mixed with thepellet and the mixture was incubated at 60° C. for 15 min to hybridizethe probe to the captured strand. Chemiluminescence from the bounddetection probes was detected substantially as described above (0.2 mlof selection reagent, pH 9.2, added to each mixture, incubated at 60° C.for 7 min, and Detect Reagents I and II added sequentially to producechemiluminescence detected in a luminometer for 5 sec). A backgroundcontrol that contained no target was treated similarly. Results of thesetests are shown in Table 1, reported as net RLU (background RLUsubtracted from each total detected RLU) and the percentage of initialtarget that was captured for each reaction. The results show that thepresence of 2.38 M and 3.18 M imidazole in the reaction mixtures greatlyincreased the target capture from the sample compared to reactions thatincluded lesser amounts or no imidazole in the same conditions.

TABLE 1 Capture of a Target Strand from dsDNA at Different ImidizoleConcentrations Imidazole (M) Detected Captured Target (RLU) CapturedTarget (%) 0 442 0.6 0.24 446 0.6 0.48 574 0.76 0.96 607 0.81 1.43 10101.34 1.91 2342 3.1 2.38 17621 24 3.18 50109 67

Additional tests were performed using the same conditions except thatthe target capture mixtures contained 0 to 3.4 M imidazole. Results areshown in Table 2, which show that about a third or more of the initialtarget was captured from reactions incubated at 60° C. that contained2.6 M to 3.4 M imidazole, and 65% or more of the target was capturedfrom mixtures that contained 3 M to 3.4 M imidazole.

TABLE 2 Capture of a Target Strand from dsDNA Imidazole Conc. (M) NetRLU Detected % Target Captured 0 688 0.92 1.5 1957 2.6 2.0 5283 7.0 2.210856 14.5 2.4 15812 21.0 2.6 24366 32.5 2.8 36872 49.2 3.0 51127 68.23.2 56916 76.0 3.4 48971 65.2

Example 3 Target Capture of a Target Strand Present in Synthetic dsDNA

Additional tests were performed using conditions substantially asdescribed in Example 2, but using different synthetic versions of thecapture probe that contained LNA or DNA residues in the 5′target-specific region (nt 1-25 of SEQ ID NO:3). Each target capturereaction included 20 fmoles of the dsDNA target in 0.2 ml of sampletransport reagent to which was added 0.2 ml of TCR(C-type) containingfrom 0 to 4.2 M imidazole, 0.1 ml of TCR containing 50 μg of magneticparticles (Sera-Mag™) with covalently attached dT₁₄ oligomers, and 20pmoles of capture probe that contained LNA or DNA residues in the 5′target-specific sequence. Reactions were incubated at 60° C. for 15 min,then at RT for 30 min, and treated as described above for capture,washing, and detection steps. Controls were treated similarly butcontained no target (background control) or contained no capture probe(net RLU: 92). Results of these tests are shown in Table 3, reported asnet RLU (background RLU subtracted from total detected RLU) and thepercentage of target captured for each reaction condition. The resultsshow that 3.5 M and 4.2 M imidazole present in the reaction mixturesincreased target capture efficiency compared to reactions that contained2.1 M imidazole. Increased target capture efficiency was seen for boththe LNA and DNA capture probes, with more target captured with the LNAprobe compared to the DNA probe.

TABLE 3 Capture of a Target Strand from dsDNA Using LNA and DNA ProbesCapture Imidazole Detected % Target Probe Conc. (M) Net RLU Captured DNA2.1 6781 4.4 3.5 92189 61 4.2 33378 22 LNA 2.1 8101 5.4 3.5 100277 66.44.2 43007 28.5

Additional target capture assays were performed by using substantiallythe same conditions described above, but using 3.2 M imidazole in thereaction mixtures and capture probes that contained different LNAportions were compared to a DNA capture probes of the same sequence. Inthose tests, a capture probe that was completely or partially LNA in the25-nt target-specific region was more efficient for target capture thanthe DNA capture probe (64-65% compared to 57.4% for the DNA probe). Acapture probe that was LNA in the target-specific region and part of thetail region (10 of 30-nt polyA) further increased target captureefficiency (86%) compared to the DNA probe (57.4%).

Using substantially the same target capture conditions with or without3.2 M imidazole in the reactions, two different detection probesspecific for the target strand (SEQ ID NO:1) were used to determinewhether the partially dsDNA target (SEQ ID NO:1 hybridized to SEQ IDNO:2) was denatured during the target capture procedure. One detectionprobe (SEQ ID NO:4, AE labeled at nt 18-19) was specific for a targetsequence at the 5′ end of the target strand, i.e., the portion of thepartially dsDNA that is single-stranded under all conditions. The seconddetection probe (SEQ ID NO:5, AE labeled at nt 18-19) was specific for atarget sequence at the 3′ end of the target strand, i.e., in the portionof the partially dsDNA that is double-stranded when added to the targetcapture mixture. The target capture reaction mixtures containing eitherthe LNA (nt 1-25) capture probe or the DNA capture probe were incubatedat 60° C. for 20 min, then at RT for 20 min, and treated as describedabove for magnetic capture, washing, and detection steps using separatealiquots for the two detection probes. For comparison, a DNA strand ofSEQ ID NO:1 was detected with each detection probe to determine thesignal for a completely single-stranded target. Without imidazole in thetarget capture reactions, capture of the target strand from thepartially dsDNA target was relatively inefficient (3.8-9.2%), whereaswith imidazole in the target capture reactions, capture of the targetstrand was efficient (79-100%). When imidazole was in the target capturereactions, the detection signals from both of the detection probes weresimilar, i.e., for a target capture mixture following capture, RLUdetected using the 3′ detection probe was similar to the RLU detectedusing the 5′ detection probe. These results show that captured strandwas single stranded from mixtures that included imidazole and wereincubated at 60° C.

Example 4 Target Capture of HBV Subtype B with Imidazole and 95° C.Incubation

This example shows that imidazole present in the target capture reagent(TCR) improved sensitivity of HBV subtype B detection in an assay formatthat combines target capture, in vitro amplification of HBV sequences(TMA), and detection of the amplified sequence by using chemiluminescentlabeled probes (U.S. Pat. No. 5,790,219, McDonough et al., US Publ. No.20040029111, Linnen et al.; PROCLEIX® Ultrio Hepatitis B Virus (HBV)Discriminatory Assay (dHBV), Chiron Corp., Emoryville, Calif.). Thisexample shows that imidazole present during target capture, inconjunction with a high temperature incubation, significantly improvedassay sensitivity. These conditions were tested using samples from aclinical serum panel known to contain HBV subtype B (used at a 1:3dilution). Twenty replicates were tested for each experimental conditionto determine the effect of the target capture modifications againstcontrol assay conditions that used the standard target capture processfollowed by the same amplification and detection assay steps. Negativecontrols (7 replicates) contained normal serum (no HBV) and were treatedidentically. Assays were conducted using the supplier's instructions(PROCLEIX® Ultrio Hepatitis B Virus (HBV) Discriminatory Assay (dHBV)package insert IN0142 Rev. 1 and 10-01-07-271 Rev. C.1), summarized asfollows. Imidazole (crystalline, FW 68.08) was added directly intostandard target capture reagent (TCR) to a final concentration of 1.7 Mor 2.7 M. Control sample tubes contained 500 μl of sample and 400 μl ofTCR without imidazole. In the modified target capture method, tubesreceived an additional 400 μl of TCR containing imidazole to a finalconcentration of 1.7 M or 2.7 M in 800 μl of TCR. Target capture tubesthat did not receive a 95° incubation step were sealed, mixed, held atRT for 15 min, incubated at 60° C. for 20 min, and then at RT for 15 minbefore separation of the hybridization complexes on the magneticsupports. Target capture tubes that received a 95° C. incubation stepwere sealed, mixed, incubated at 95° C. for 15 min without agitation,then incubated at 60° C. for 20 min, and RT for 15 min. Tubes weretreated to separate magnetically the particles with captured nucleicacids, wash the captured nucleic acids on the particles, and thenhandled to perform the in vitro amplification and detection of HBVsequences. Briefly, captured HBV nucleic acids were amplified forspecific sequences by using a combination of HBV specific primers andTMA to produce amplified products that were detected by hybridizationwith labeled probes that bind specifically to the amplified HBVsequences (US Publ. No. 20040029111). Signals from the labels associatedwith bound probes were produced and detected as chemiluminescence (RLU)in a homogeneous detection assay. Results shown in Table 4.

TABLE 4 HBV Detection Using Different Target Capture Conditions SampleInitial TCR ± % Mean (No. Tested) Heating Step Imidazole Positive RLUNegative control none 400 μl TCR 0 748 (n = 7) HBV-B Panel none 400 μlTCR 30 830,181 (n = 20) HBV-B Panel none 800 μl TCR + 50 803,816 (n =20) 1.7M imidazole HBV-B Panel none 800 μl TCR + 25 852,630 (n = 20)2.7M imidazole Negative control 95° C. 400 μl TCR 0 1,080 (n = 7) for 15min HBV-B Panel 95° C. 400 μl TCR 44 1,078,457 (n = 20) for 15 min HBV-BPanel 95° C. 800 μl TCR + 100 971,403 (n = 20) for 15 min 1.7M imidazoleHBV-B Panel 95° C. 800 μl TCR + 100 1,046,232 (n = 20) for 15 min 2.7Mimidazole

The results show that the target capture process that included imidazolein the reactions and a 95° C. incubation improved detection of HBV inthe assays compared to assays that used a target capture procedure thatdid not include imidazole in the reaction mixture and 95° C. incubation.

Example 5 Efficient Target Capture of HBV Subtypes A, B and C Over aRange of Temperatures

This example shows that the efficient target capture method described inExample 4 improved assay sensitivity for HBV subtypes A and C, and theimproved target capture efficiency associated with the presence ofimidazole occurs over a temperature range. The target capture proceduresconducted with use of imidazole at various temperatures were testedusing samples of HBV clinical serum panels for subtypes A, B and C.Thirty replicates of each subtype (at 1:3 dilution) were tested todetermine the effect on target capture of TCR containing 2.7 M imidazoleincubated for 12 min at 64° C., 75° C., 85° C., 90° C. or 95° C. Assayswere performed substantially as described in Example 4, but at differenttemperatures. Some of the results of the tests are shown in FIG. 1,including those for a control in which TCR contained no imidazole andincubation was at RT (“Room Temp.—No Imidazole”). The results show theeffect of the presence of 2.7 M imidazole during target capture usingthree incubation temperatures (64° C., 85° C., and 95° C.) for threedifferent HBV nucleic acid targets: subtype A (medium shaded), subtype B(dark shaded), and subtype C (light shaded). The percentage of positiveresults (y-axis) are shown for samples tested using the differentconditions (x-axis), for assays performed on separate days (Day 1 andDay 2). Numbers above the bars indicate the percent positive detectionof HBV for the tested samples. From these and other results, theefficient target capture method that included imidazole was shown toimprove assay sensitivity over a broad temperature range for HBVsubtypes A and C (from about 75° C. to 95° C.), but over a narrowertemperature range for HBV subtype B (from about 90° C. to 95° C.).

Example 6 Target Capture of a Partial dsDNA/RNA Virus, HBV Subtype B

This example shows that the efficient target capture method thatcombines imidazole in the reaction mixture and 95° C. incubation can beperformed for a shorter time than typically required for nucleic aciddenaturation at high temperature. That is, the synergistic effectachieved by using 2.7 M imidazole and 95° C. incubation was demonstratedin a short time range.

Assays were performed substantially as described in Examples 4 and 5,but using different incubation times. The target capture process thatincludes imidazole in TCR was tested on samples of a HBV subtype Bclinical serum panel. Twenty replicates (at 1:10 dilution) were testedusing TCR containing 2.7 M imidazole in mixtures incubated at 95° C. for1, 3, 5 and 7 min. The TCR also contained an internal control nucleicacid unrelated to the target nucleic acid. The tests were compared tosimilar tests performed by using the standard target capture process.For both sets of tests, captured nucleic acids were used inamplification and detection steps performed substantially as describedin Examples 4 and 5. Results shown in FIG. 2 demonstrate that a 95° C.incubation step of as short at 3 min when imidazole is present in thetarget capture mixture improved assay sensitivity significantly comparedto the same assay performed by using the standard target capture methodthat does not include imidazole in the mixture or a 95° C. incubation.FIG. 2 shows the increase in percent positive detection (y-axis andnumber over each bar) for the conditions tested (x-axis). The resultsshow that incubation for 3 min or more at 95° C. is adequate to producethe synergistic effect that increases target capture efficiency in thepresence of imidazole, which results in increased assay sensitivity.

Example 7 Target Capture Performed with Different Chemical Denaturants

This example demonstrates the effects on detection of a DNA target whena target capture process includes target capture in the presence of 8 Murea or 2 M Imidazole and incubation at 95° C. The target polynucleotidewas a sequence specific to HBV genotype C. Twenty replicate samples weretested for each condition in assays performed substantially as describedExamples 5 and 6, i.e., different target capture processes followed byidentical in vitro amplification of the captured nucleic acids anddetection of amplified sequences. These assays compared results obtainedwhen samples were subjected to target capture with the followingvariables: (1) TCR plus 8 M urea, (2) 95° C. incubation, (3) TCR plus 8M urea plus 95° C. incubation, and (4) TCR plus 2 M imidazole plus 95°C. incubation, all compared to a standard target capture process thatdoes not include urea or imidazole in reactions or 95° C. incubation.Briefly, the assay protocol was as follows. Three TCR versions were madeand then used immediately thereafter: TCR with 2 M imidazole, TCR with 8M urea, and standard TCR (no urea or imidazole). Each reaction tubecontained 400 μl of one TCR version, into which was added 500 μl of HBVgenotype C (HBV-C) or 500 μl of normal serum (negative controls). Tubeswere sealed and mixed. Some tubes were incubated at 95° C. for 10 min,followed by RT for 5 min, whereas other tubes remained at RT for 15 min(i.e., no 95° C. incubation). Then, the standard target capture protocolwas followed as described above (60° C. for 20 min, RT for 15 min,magnetic separation of particles with captured nucleic acids, and washstep). Captured target polynucleotides were amplified by using TMA withprimers specific for HBV and amplified sequences were detected by usingAE-labeled probes specific for the amplified sequence to producechemiluminescence which was detected (RLU) and used to determine whetherthe assays produced positive or negative results (RLU greater than50,000 were considered positive). Results of the assays are summarizedin Table 5.

TABLE 5 HBV Type C Detection in Assays Using Different Target CaptureConditions 95° C. % Positive Samples TCR Incubation Results HBV-CStandard No 35% HBV-C +2.0M imidazole Yes 100% HBV-C +8.0M urea Yes 100%HBV-C +8.0M urea No 25% HBV-C Standard Yes 25% Negative ControlsStandard No 0%

The results show that the presence 2 M imidazole or 8 M urea in thetarget capture reaction mixture combined with 95° C. incubation greatlyincreased sensitivity of the assay to detect the HBV target. Targetcapture conditions that included a denaturant chemical (imidazole orurea) in the reaction mixtures incubated at 95° C. demonstrated thesynergistic effect of these conditions, compared to target capturemixtures that used only the 95° C. incubation or that included urea butwithout the 95° C. incubation.

1. A method for isolating a target nucleic acid of interest from asample, comprising: mixing a sample containing a target nucleic acidwith a capture probe that hybridizes specifically to a target sequencein the target nucleic acid in a solution phase that contains adenaturant chemical and an immobilized probe that binds specifically tothe capture probe, to provide a reaction mixture, incubating thereaction mixture at a first temperature in a range of about 60° C. to95° C. for about 15 minutes or less, incubating the reaction mixture ata second temperature in a range of about 25° C. to 42° C. for about 20minutes or less, thereby forming a hybridization complex made up of thecapture probe hybridized specifically to the target nucleic acid and theimmobilized probe bound specifically to the capture probe, wherein thehybridization complex is attached to a support via the immobilizedprobe, and separating the hybridization complex attached to the supportfrom other sample components.
 2. The method of claim 1, wherein thedenaturant chemical is 8 M urea and incubating at the first temperatureis about 95° C. for about 10 minutes or less.
 3. The method of claim 1,wherein the denaturant chemical is imidazole at a concentration from 0.5M to 4.2 M, and incubating at the first temperature is about 60° C. forabout 1 to 15 minutes.
 4. The method of claim 1, wherein the denaturantchemical is imidazole at a concentration from 3.0 M to 3.5 M, andincubating at the first temperature is at 60° C. for about 1 to 15minutes.
 5. The method of claim 1, wherein the denaturant chemical isimidazole at a concentration from 2.0 M to 2.7 M and incubating at thefirst temperature is about 90° C. to 95° C. for about 3 to 10 minutes.6. The method of claim 1, wherein the denaturant chemical is imidazoleat 2.7 M, incubating at the first temperature is about 75° C. to 95° C.for about 3 to 15 minutes, and the method further includes incubatingthe reaction mixture at about 60° C. for about 20 minutes between thefirst and second incubating steps.
 7. The method of claim 6, whereinincubating at the first temperature is about 95° C. for about 3 to 15minutes.
 8. The method of claim 1, wherein the target nucleic acid iscompletely or partially double-stranded nucleic acid, or a nucleic acidthat includes other secondary or tertiary structure.
 9. The method ofclaim 1, wherein the capture probe is made up of a target-specificsequence that binds to the target nucleic acid and a tail region thatbinds to the immobilized probe via a specific binding partner.
 10. Themethod of claim 9, wherein the tail region binds to the immobilizedprobe by hybridizing specifically to a complementary sequence of theimmobilized probe.
 11. The method of claim 1, further comprising a stepof detecting the target nucleic acid or an in vitro amplificationproduct made from the target nucleic acid after separating thehybridization complex attached to the support from other samplecomponents.
 12. A method for isolating a target nucleic acid of interestfrom a sample, comprising: mixing a sample containing a target nucleicacid with a capture probe that hybridizes specifically to a targetsequence in the target nucleic acid in a solution phase that contains adenaturant chemical and an immobilized probe that binds specifically tothe capture probe, to provide a reaction mixture, incubating thereaction mixture at about 25° C. for about 1 minute to 60 minutes,thereby forming a hybridization complex made up of the capture probehybridized specifically to the target nucleic acid and the immobilizedprobe bound specifically to the capture probe, wherein the hybridizationcomplex is attached to a support via the immobilized probe, andseparating the hybridization complex attached to the support from othersample components.
 13. The method of claim 12, wherein the denaturantchemical is urea at a concentration of about 1 M.
 14. The method ofclaim 12, wherein the denaturant chemical is imidazole at aconcentration between 0.05 M and 0.5 M and the incubating step is forabout 2 to 30 minutes.
 15. The method of claim 12, wherein thedenaturant chemical is imidazole at a concentration of about 0.5 M andthe incubating step is for about 15 minutes.
 16. A composition forspecific capture of a target nucleic acid, comprising at least onetarget nucleic acid, at least one capture probe that hybridizesspecifically to a target sequence in the target nucleic acid, animmobilized probe that binds specifically to the capture probe, and asolution phase hybridization mixture that contains imidazole at aconcentration from 0.05 M to 4.2 M or urea at a concentration from 1 to8 M.
 17. The composition of claim 16, wherein the solution phasehybridization mixture contains imidazole at a concentration of from 0.05to 0.5 M.
 18. The composition of claim 16, wherein the solution phasehybridization mixture contains imidazole at a concentration from 1.7 Mto 3.5 M.
 19. The composition of claim 16, wherein the solution phasehybridization mixture contains imidazole at a concentration from 2.0 Mto 2.7 M.
 20. The composition of claim 16, wherein the compositionincludes a first capture probe that hybridizes specifically to a firsttarget sequence and a second capture probe that hybridizes specificallyto a second target sequence which is different from the first targetsequence.